Neutralizing monoclonal antibody in human adenovirus 7 and preparation method and application thereof

A monoclonal antibody, adenovirus technology, applied in microorganism-based methods, biochemical equipment and methods, antibodies, etc., can solve the difficulty of neutralizing monoclonal antibodies, difficult to show spatial conformation, and the virus cannot be cultured in cell lines. Yield and other problems to achieve the effect of enhancing the immune response effect

Active Publication Date: 2014-10-08
广州瑞发一号健康投资中心(有限合伙)
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

We know that some viruses cannot be cultured in cell lines or have low yields in vitro
Therefore, it is very difficult to prepare neutralizing mAbs against these pathogens by immunizing with pathogen particles
Epitope peptide-based immunization methods are one way to solve this problem, but the neutralizing epitopes of many pathogens are conformational epitopes, and it is difficult for simple peptides and peptides presented or displayed by the above methods to exhibit spatial conformation

Method used

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  • Neutralizing monoclonal antibody in human adenovirus 7 and preparation method and application thereof
  • Neutralizing monoclonal antibody in human adenovirus 7 and preparation method and application thereof
  • Neutralizing monoclonal antibody in human adenovirus 7 and preparation method and application thereof

Examples

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Embodiment 1

[0044] Example 1: Construction of type 3 adenovirus vector Ad3EGFP

[0045] The adenovirus vector constructed in the present invention is based on the deletion of the human HAdv3 E3 region and the expression of the enhanced green fluorescent protein, and can express the recombinant adenovirus of the HAdv3 genome and the enhanced green fluorescent protein. The specific construction steps are as follows: after the HAdv3GZ01 virus strain was cultured in HEp-2 cells, the virus genome was extracted, and homologously recombined with the shuttle plasmid pBRALR in BJ5183 bacteria to obtain the plasmid pBRAdV with the full genome of the HAdv3GZ01 virus; after RsrII digestion and linearization of the plasmid pBRAdV Homologous recombination with the shuttle plasmid pSKE3LCMVeGFP-SV40R in BJ5183 bacteria, the recombinant plasmid pBRAd△E3GFP inserted into the EGFP gene expression cassette was obtained, and the recombinant adenovirus rAd△E3GFP was rescued after the plasmid was transfected in...

Embodiment 2

[0047] Example 2: Construction of immunogen rAdMHE3

[0048] The hexon hypervariable region 5 (HVR5) of Ad3EGFP ( 264 FDGRDAVAGALAPEIV 279 ) (the amino acid sequence of the 5th hypervariable region of the hexon of the human type 3 adenovirus vector is shown in SEQ NO: 2, and the sequence is located at the 264th to the 264th position of the hexon of the human type 3 adenovirus vector 279 bits) replaced by HAdv7's HVR5 ( 255 FDGREAADAFSPEIV 269 ) (the amino acid sequence of the 5th hypervariable region of the hexon of human adenovirus type 7 is shown in SEQNO: 1, and the sequence is located at the 255th to 269th position of the hexon of the human adenovirus type 7) , by-pass PCR to amplify the hexon gene of human type 3 cells mutated in the HVR5 region, digest and connect to the shuttle vector pBRLR, and homologously recombine with the vector pBRAd△E3GFP in BJ5183 bacteria to obtain the hexon-mutated recombinant adenoviral plasmid pBRAd- MHE3, rescue of recombinant virus rAd...

Embodiment 3

[0050] Embodiment 3: Identification uses the construction of chimeric virus rAdH7R1, rAdH7R2, rAdH7R5 and rAdH7R7

[0051] First construct rAd3egf / H7 (Ad3 / H7) chimeric virus, rAd3egf / H7 (Ad3 / H7) chimeric virus is a chimeric virus produced by replacing the hexon gene of Ad3EGFP with the hexon gene of HAdv7GZ08. Construction method: PCR amplified the hexon fragment of HAdv7GZ08 virus strain, connected to the shuttle plasmid pBRALR, homologously recombined with the pBRAd△E3GFP backbone plasmid in BJ5183 to obtain the recombinant plasmid pAd3egf / H7, and rescued and recombined after transfecting 293 cells Virus rAd3egf / H7. The neutralizing antibody titer experiment was carried out on the virus-immunized mice, and the experimental results are shown in Table 1. Table 1 is the result table of the neutralizing antibody titer experiment on the adenovirus-immunized mice. It can be seen from the data in Table 1 that the hexon in the virion is the hexon protein of adenovirus type 7, and t...

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Abstract

The invention discloses a method for preparing a neutralizing monoclonal antibody in a human adenovirus 7. The hexon fifth hypervariable region of a human adenovirus 3 vector is substituted by a hexon fifth hypervariable region gene of the human adenovirus 7 to construct a chimeric virus which is taken as an immunogen. A murine human adenovirus 7 neutralizing monoclonal antibody and a hybridoma cell strain 5MH5 / 3G5 prepared by using the method are further provided. A humanized and chimeric monoclonal antibody prepared by using the method and a medicinal composition comprising the humanized or human-mouse chimeric monoclonal antibody serving as an effective component are further provided. An exogenous neutralizing epitope is embedded into the hypervariable region of the human adenovirus 3 vector to construct a recombinant adenovirus showing the exogenous neutralizing epitope. By using the recombinant adenovirus as an immunogen, the exogenous neutralizing epitope can be shown effectively, the immune response effect is enhanced, and the research of a monoclonal antibody for treating infectious diseases is facilitated.

Description

technical field [0001] The invention belongs to the technical field of monoclonal antibody preparation, and in particular relates to a neutralizing monoclonal antibody of human type 7 adenovirus and its preparation method and application. Background technique [0002] The use of epitope peptide immunization to prepare monoclonal antibodies, especially therapeutic monoclonal antibodies, is an attractive approach. Epitope peptides represent the smallest immunogenic unit of a protein antigen. Therefore, epitope peptide immunization can precisely position the immune response, and can well control the generation of the desired immune response. However, the application of epitope peptide immunization to prepare monoclonal antibodies usually has a very low positive rate. Because simple peptides usually have poor immunogenicity, cannot present a good spatial conformation, and cannot effectively activate T cells and immune memory cells. Because of this, many methods to improve the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N5/20A61K39/42A61P31/20C12R1/91
Inventor 周荣李晨阳刘铭龙田新贵
Owner 广州瑞发一号健康投资中心(有限合伙)
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